Epha2 antigen t epitopes

ABSTRACT

The invention concerns peptides constituting T epitopes of EphA antigen, exhibited by MHC I. Said peptides are useful in particular in antitumoral immunotherapy.

The present invention relates to peptides derived from the EphA2 proteinand to their use in antitumor immunotherapy.

Peptide immunization or immunotherapy is a therapeutic approach which iscurrently the subject of a great deal of interest in the context of theprevention or treatment of cancer. The principle thereof is based onimmunization with peptides which reproduce T epitopes of tumor antigensrecognized by cytotoxic T lymphocytes (CTLs), which play a major role inthe elimination of cancer cells expressing these antigens at theirsurface.

It will be recalled that CTLs do not recognize whole protein antigens,but peptide fragments thereof, presented by major histocompatibilitycomplex (MHC) molecules expressed at the surface of various cells. It isthese peptide fragments which constitute the T epitopes.

The presentation of these peptides is the result of a complex process,called “antigen processing”, which involves 3 main steps:

-   -   cytosolic degradation of the antigens by a multienzyme complex        called proteasome;    -   translocation of the peptides derived from this degradation into        the endoplasmic reticulum (ER) by TAP transporters;    -   association of these peptides with the MHC so as to form stable        peptide/MHC complexes which will be exported to the cell        surface.

The presentation of T epitopes at the cell surface depends in particularon the stability of the antigenic protein in the cytosol, on the sitesand on the frequency of the cleavages carried out by the proteasome, onthe efficiency of translocation into the ER by the TAP transporters, andon the ability of the peptides to bind to the MHC molecules and to formstable peptide/MHC complexes.

The epitopes presented by the major histocompatibility complex class I(MHC I) generally have 8 to 11 amino acids, and are recognized by CD8+ Tcells, which represent the major component of the cytotoxic response.The epitopes presented by the major histocompatibility complex class II(MHC II) generally have 13 to 18 amino acids and are recognized by CD4+T cells.

The identification of these epitopes, and in particular (given theessential role of the CD8+ response in cytotoxicity) of those presentedby MHC I, therefore constitutes an essential step in the development ofantitumor immunotherapy compositions.

Many tumor antigens capable of inducing a CTL response are known at thecurrent time. Some of the T epitopes of these antigens have beenidentified, and the effectiveness of vaccines based on peptides whichreproduce these T epitopes has been shown in many cases. However, theexpression of the majority of these antigens is restricted to certainhistological types of tumors, which limits their clinical use. It istherefore desirable to identify other tumor antigens expressed by alarge number of tumors of varied origin, and which are also capable ofinducing an antitumor cytotoxic immune response.

The EphA2 receptor, previously called ECK (Lindberg and Hunter, Molec.Cell. Biol. 10, 6316-6324, 1990), is a membrane receptor which hastyrosine kinase activity. The sequence of the human EphA2 receptor isrepresented in FIG. 1 (SEQ ID NO: 1). This receptor comprises anextracellular domain of 534 amino acids, a transmembrane domain of 24amino acids and a cytoplasmic domain of 418 amino acids which containstyrosine kinase domain. This receptor is overexpressed in several typesof cancer, such as colon cancer, breast cancer, prostate cancer, lungcancer, stomach cancer, esophageal cancer and metastatic melanoma, butis not overexpressed in non-cancerous lesions in these same tissues(Rosenberg et al. Am. J. Physiol. 273, 824, 1997; Zelinski et al. CancerRes. 61, 2301, 2001; Nemoto et al. Pathobiology 65, 195, 1997; Easty etal. Int. J. Cancer 60, 129, 1995; Walker Daniel et al. Prostate 41, 275,1999). It has been observed that the overexpression of EphA2 is linkedto malignant transformation and facilitates the metastatic progressionof tumors. In addition, EphA2 plays an important role in tumorneovascularization (Ogawa et al. Oncogene 19, 6043, 2000).

Due to its overexpression in many types of tumors, and to itsinvolvement in malignant transformation and in tumor angiogenesis, ithas been proposed to use EphA2 as a target for antitumor treatments.Thus, PCT application WO 01/121172 proposes the use of antibodiesdirected against B epitopes carried by the extracellular domain of theEphA2 receptor for passive antitumor immunotherapy.

However, it was not known, until now, whether EphA2 could be effectivelyprocessed so as to generate T epitopes capable of inducing an antitumorcytotoxic response. A fortiori, no T epitope of this antigen had beenidentified.

The inventors have now identified, in EphA2, immunogenic peptidespresented by MHC I which induce cytotoxic T lymphocytes capable oflysing tumor cells expressing EphA2.

Consequently, a subject of the present invention is an immunogenicpeptide constituting a T epitope presented by MHC I, characterized inthat it consists of a fragment of 8 to 11 consecutive amino acids of theEphA2 antigen.

In the context of the disclosure of the present invention, the term“immunogenic peptide” is intended to mean a peptide capable of inducinga specific CTL response against the EphA2 antigen.

Peptides in accordance with the invention can be obtained from the EphA2antigen in various ways. For example, it is known that peptides capableof forming a complex with a given MHC I allele have in common thepresence, at certain positions, of specific amino acid residues called“anchor residues”. Specific anchor motifs involving amino acids called“primary anchor residues” have thus been defined for the various MHC Ialleles. It has also been shown that residues located outside theprimary anchor motifs (secondary anchor residues) can exert a favorableor unfavorable effect on the affinity of the peptide for MHC.

The choice of the peptide sequences capable of constituting epitopespresented by a given MHC I allele can be made, conventionally, byanalyzing the peptide sequence of the EphA2 antigen in order to selectthe peptides which have all or part of the primary anchor motifcorresponding to this allele. Various databases which list the knownanchor motifs are available: by way of examples, mention will be made ofthe SYFPEITHI base (http://www.uni-tuebingen.de/uni/kxi/; Rammensee etal., Immunogenetics, 50, 213-219, 1999), or the BIMAS base(http://bimas.dcrt.nih.gov/molbio/hla bind; Parker et al., J. immunol.152, 163, 1994).

Generally, the binding affinity of the peptides thus identified for theallele of the concerned will then be determined, as will the stabilityof the peptide/MHC I molecule complex. In fact, non-immunogenic peptidesmost commonly exhibit weak affinity for MHC I molecules, and/or formwith them a relatively unstable complex. Methods for determining theaffinity of the peptide for the MHC I molecule and the stability of thecomplex formed are known per se. Mention will be made, for example, ofthat described by Firat et al. (Eur. J. Immunol., 29, 3112, 1999).

The affinity of a peptide for an MHC I molecule is most commonly definedwith respect to that of a reference peptide (for example IVGAETFYV (SEQID NO: 2) for HLA-A*0201 or RPHERNGFTV (SEQ ID NO: 3) for HLA-B*0702),in the form of relative affinity. The relative affinity is defined asthe ratio of the concentration of the peptide tested to theconcentration of the reference peptide which allows the formation, underthe same conditions, of the same amount of peptide/MHC I moleculecomplex. The higher the relative affinity, the lower the bindingaffinity of the peptide for the MHC I molecule.

The stability of the peptide/MHC I molecule complex is often defined bythe DC₅₀, which represents the time required for the dissociation of 50%of the complexes formed.

For example, in the case of potentially immunogenic peptides presentedby HLA-A*0201, the relative affinity is generally less than 5 and theDC₅₀ greater than 2 hours.

The immunogenicity of the potentially immunogenic peptides thus detectedcan be verified, for example by means of conventional methods fordetermining the ability of this peptide to generate, in vivo, ex vivo orin vitro, a specific CTL response with respect to target cells loadedwith this peptide, or expressing the EphA2 antigen from which it isderived.

The peptides which have a weak affinity for the MHC I moleculeconcerned, and/or which form with the latter a relatively unstablecomplex, generally have a weak immunogenicity. However, these peptidesmay be of therapeutic interest insofar as it appears that low-affinityepitopes do not contribute, or only slightly contribute, to theestablishing of tolerance phenomena, which constitute one of the mainpitfalls of antitumor immunization.

In this case, it is possible to prepare variant peptides which havegreater immunogenicity, by substitution of one or more of the aminoacids of the native peptide with one or more amino acids favorable tothe affinity for the MHC I molecule concerned and/or to the stability ofthe peptide/MHC I molecule complex.

These variant peptides are also part of the subject of the presentinvention.

Amino acids that are favorable to the affinity for a given MHC Imolecule and/or to the stability of the peptide/MHC I molecule complexmay, for example, consist of anchor residues, and in particular thesecondary anchor residues, known for the MHC I molecule concerned. Theseanchor residues can be readily identified by consulting the availabledatabases, such as those mentioned above.

By way of example of substitution making it possible to increase theimmunogenicity of a peptide presented by an MHC I molecule, and inparticular by HLA-A*0201, mention will be made of the substitution ofthe N-terminal amino acid of said peptide with a tyrosine, as describedin PCT application WO 02/08716.

The affinity of a variant peptide for the MHC I molecule concerned, andalso its immunogenicity, can then be verified as indicated above for thenative peptides.

By way of nonlimiting example of implementation of the presentinvention, the inventors have identified five peptides, hereinafterreferred to as p58, p61, p546, p550 and p883, presented by HLA-A*0201.

The sequences (1-letter code) of these peptides are as follows: p58:IMNDMPIYM; (SEQ ID NO: 4) p61: DMPIYMYSV; (SEQ ID NO: 5) p546:VLLLVLAGV; (SEQ ID NO: 6) p550: VLAGVGFFI; (SEQ ID NO: 7) p883:TLADFDPRV. (SEQ ID NO: 8)

The inventors have also obtained, from the p61 peptide, which exhibitsonly weak affinity for HLA-A*0201 and weak immunogenicity, animmunogenic peptide, hereinafter referred to as p61Y, of sequenceYMPIYMYSV (SEQ ID NO: 9), resulting from the substitution of theN-terminal residue of p61 with a tyrosine residue.

These peptides are capable of inducing a specific CTL response withrespect to HLA-A*0201 cells expressing EphA2. They induce in particulara cytotoxic response with respect to HLA-A*0201 tumor cells derived fromtumors of varied types.

A subject of the present invention is also compositions comprising atleast one immunogenic peptide in accordance with the invention, or anucleic acid molecule encoding said peptide.

They may be multiepitope compositions capable of generating apolyspecific CTL response, and which, with the same, also comprise oneor more other immunogenic epitope(s). These other epitopes may bederived from EphA2 or from one or more other antigens.

These multiepitope compositions in accordance with the invention maycomprise, so that they can be widely used on a population whoseindividuals carry different HLA alleles, epitopes presented by variousMHC I molecules. They may also comprise, in addition, at least oneepitope presented by an MHC II molecule and capable of inducing aT-helper response.

According to a preferred embodiment of a composition in accordance withthe invention, it comprises at least one chimeric polypeptide comprisingone or more copies of an immunogenic peptide in accordance with theinvention. In the case of a multiepitope composition, said chimericpolypeptide also comprises one or more copies of at least one otherimmunogenic epitope.

Such a chimeric polypeptide can be readily obtained by methods known perse, and in particular by conventional recombinant DNA techniques.

A subject of the present invention is also the nucleic acid moleculesencoding an immunogenic peptide or a chimeric polypeptide in accordancewith the invention.

A subject of the present invention is also the use of an immunogenicpeptide epitope, of a composition or of a nucleic acid molecule inaccordance with the invention, for obtaining a medicinal product, and inparticular a medicinal product intended for antitumor immunotherapy, andin particular for the treatment of tumors expressing EphA2.

This encompasses a large variety of tumors, among which mention will inparticular be made of colon tumors, breast tumors, prostate tumors, lungtumors, stomach tumors, kidney tumors and esophageal tumors.

The p58, p61, p546, p550, p883 and p61Y peptides can in particular beused for obtaining medicinal products intended for the treatment ofHLA-A*0201 patients.

The present invention also encompasses the medicinal productscomprising, as active principle, at least one immunogenic peptide, onecomposition or one nucleic acid molecule in accordance with theinvention.

According to a preferred embodiment of the present invention, saidmedicinal products are vaccines.

Medicinal products in accordance with the invention can also comprisethe usual excipients, and also adjuvants conventionally used inimmunotherapy and which make it possible, for example, to promote theadministration of the active principle, to stabilize it, to increase itsimmunogenicity, etc.

The present invention will be understood more thoroughly from thefurther description which follows, which refers to nonlimiting examplesillustrating the induction of an antitumor cytotoxic response bypeptides in accordance with the invention derived from the EphA2antigen.

EXAMPLE 1 Identification of EphA2 Epitopes Presented by the HLA-8*0201Molecule

The amino acid sequence of the EphA2 protein was analyzed by means ofthe BIMAS program (Parker et al., J. Immunol. 152, 163, 1994), in orderto identify peptides potentially capable of binding to HLA-A*0201. Amongthe potential epitopes identified, the following five peptides: p58:IMNDMPIYM; (SEQ ID NO: 4) p61: DMPIYMYSV; (SEQ ID NO: 5) p546:VLLLVLAGV; (SEQ ID NO: 6) p550: VLAGVGFFI; (SEQ ID NO: 7) p883:TLADFDPRV; (SEQ ID NO: 8)

-   -   were selected.

The peptides corresponding to these sequences were synthesized bySYNT:EM (Nimes, France). The purity (>85%) is monitored by reverse-phasehigh performance liquid chromatography. The peptides are lyophilized andthen dissolved in DMSO at 10 mg/ml and stored at −80° C.

The immunogenicity of these peptides was evaluated by measuring theiraffinity for HLA-A*0201. This is defined by two parameters: the relativeaffinity (RA) which reflects the ability of the peptides to bind toHLA-A*0201, and the rate of dissociation of the HLA-A*0201/peptidecomplexes (DC₅₀), which reflects their stability. The high-affinitypeptides (RA<5 and DC₅₀>2 hrs) are potentially immunogenic, unlike thelow-affinity peptides (RA>5 and DC₅₀<2 hrs).

Relative Affinity:

Human T2 cells (Firat et al., Eur. J. Immunol., 29, 3112, 1999) (3×10⁵cells/ml), which are deficient in TAP transporters, are incubated at 37°C. for 16 hours with various concentrations (100 μM, 10 μM, 1 μM, 0.1μM) of each test peptide, in serum-free RPMI 1640 medium supplementedwith 100 ng/ml of human β2-microglobulin. Next, they are washed twiceand labeled with the monoclonal antibody BB7.2 (Parham et al., Hum.Immunol., 3, 4, 277-299, 1981) which is specific for the HLA-A*0201molecule, and then with a goat anti-mouse Ig antibody coupled tofluorescein isothiocyanate (FITC).

These cells are then analyzed by flow cytometry. For each concentrationof peptide, the fluorescence specific for HLA-A*0201 is calculated as apercentage of the fluorescence obtained with 100 μM of a referencepeptide (HIVpol 589; IVGAETFYV; SEQ ID NO: 2). The relative affinity(RA) is defined as the ratio of the concentration of each peptide thatinduces 20% of the fluorescence obtained with 100 μM of the referencepeptide, to the concentration of the reference peptide that induces 20%of the fluorescence obtained with 100 μM of said reference peptide. Thelower the relative affinity, the stronger the binding of the peptide toHLA-A*0201. The mean RA for each peptide is determined from at leastthree independent experiments. In all the experiments, 20% of themaximum fluorescence was obtained for 1 to 3 μM of the referencepeptide.

Stability:

T2 cells (10⁶/ml) are incubated overnight at 37° C. with 100 μM of eachtest peptide, in serum-free RPMI 1640 medium supplemented with 100 ng/mlof human β2-microglobulin. Next, they are washed four times in order toremove the free peptides, incubated with BREFELDIN A (Sigma; 10 μg/ml)for one hour in order to prevent the expression at their surface ofnewly synthesized HLA-A*0201 molecules, washed, and incubated at 37° C.for 0, 2, 4, 6 or 8 hours in the presence of BREFELDIN A (0.5 μg/ml).For each incubation time, the cells are then labeled, as indicatedabove, with the BB7.2 antibody and analyzed by flow cytometry in orderto evaluate the amount of peptide/HLA-A*0201 complex present at theirsurface. This amount is evaluated by means of the formula: (meanfluorescence of the T2 cells preincubated with the peptide−meanfluorescence of the T2 cells treated under similar conditions in theabsence of peptide). The DC₅₀ (dissociation complex: DC) is defined asbeing the time (in hours) required for the loss of 50% of theHLA-A*0201/peptide complexes which are stabilized at t=0.

The results of these experiments are given in Table 1 below. TABLE 1Peptide Sequence RA DC₅₀ p58 IMNDMPIYM 1 4 p61 DMPIYMYSV >10 ND P61YYMPIYMYSV 1.5 ND p546 VLLLVLAGV 1.4 4-6 p550 VLAGVGFFI 1 4-6 p883TLADFDPRV 2.2 2-4ND: not determined

These results show that the p58, p546, p550 and p883 peptides have aconsiderable binding affinity (RA of 1 to 2.2). On the other hand, thep61 peptide has a weak affinity for HLA-A*0201 (RA>10) and shouldtherefore not be immunogenic. In order to improve the affinity of thispeptide for HLA-A*0201, the inventors substituted the aspartic acid atposition 1 with a tyrosine residue. The variant peptide p61Y obtainedexhibits a considerable binding affinity (RA=1.5) that is clearlygreater than that of the peptide from which it is derived.

The results also show that the p58, p546, p550 and p883 peptides formstable complexes with the HLA-A*0201 molecules (DC₅₀>2 h for each ofthem).

EXAMPLE 2 Immunogenicity of the P58, p61Y, p546, p550 and p883 Peptides

Induction of Specific CTLs by Immunization with the Peptides

The immunogenicity of the p58, p61Y, p546, p550 and p883 peptides wasevaluated, by generation of CTLs, on HHD transgenic mice (Pascolo etal., J. Exp. Med., 185, 2043, 1997). These mice are β2m−/−, D^(b)−/− andexpress an HLA-A*0201 single chain made up of the α1 and α2 domains ofHLA-A*0201 and of the α3 and intracellular domains of D^(b), linked viaits N-terminal end to the C-terminal end of human β2-microglobulin bymeans of a 15 amino acid peptide.

The HHD mice receive a subcutaneous injection, at the base of the tail,with 100 μg of each test peptide emulsified in incomplete Freund'sadjuvant, in the presence of 140 μg of a T-helper epitope derived fromthe HBV “core” antigen (128-140, sequence TPPAYRPPNAPIL, SEQ ID NO: 10).

After 11 days, spleen cells taken from the mice (5×10⁷ cells in 10 ml)are stimulated in vitro with the test peptide (10 μM). On the 6th day ofthe culture, the populations which respond are tested in order todetermine a specific cytotoxicity. The cells which respond arerestimulated in vitro at one-week intervals with 2×10⁷ irradiated (3000rads) HHD spleen cells and 1 to 0.1 μM of peptide in the presence of 50IU/ml of recombinant IL2 (Proleukin, Chiron Corp.)

Cytotoxicity assays are carried out 6 days after the final stimulation.

RMAS-HHD cells are used as targets to study the cytotoxicity. Thesecells are obtained by transfection of murine RMAS cells with the HHDconstruct as described by Pascolo et al. (J. Exp. Med., 185, 2043,1997).

These target cells are labeled with 100 μCi of ⁵¹Cr for 90 minutes, andthen washed three times and plated out in round-bottomed 96-well plates(3×10³ cells/well in 100 μl of RPMI 1640+3% of fetal calf serum). Theyare loaded with 1 μM of the test peptide, or of an irrelevant controlpeptide, at 37° C. for 90 minutes.

Next, 100 μl of the effector cells (effector cells/target cellratio=40/1) are added to the wells and the plates are incubated at 37°C. for 4 hours. After incubation, 100 μl of supernatant are collectedand the radioactivity is measured in a γ-counter.

The percentage specific lysis is calculated by means of the formula[(experimental ⁵¹Cr release−spontaneous ⁵¹Cr release)/(maximum ⁵¹Crrelease−spontaneous ⁵¹Cr release)]×100. In all the experiments, thespontaneous release is less than 20% of the maximum release induced with3N HCl.

The results of these experiments for the p58 and p550 peptides are givenin FIG. 2.

-   □: irrelevant peptide;-   ▪: EphA2 peptide.

These results show that immunization with the p58 or p550 peptidegenerates CTLs which kill the RMAS-HHD targets loaded with this samepeptide, but not the cells loaded with the irrelevant peptide.Equivalent results were obtained with the p61Y, p546 and p883 peptides.

CTL lines, respectively called mCTL58, mCTL61Y, mCTL546, mCTL550 andmCTL883, were established from the spleen cells of HDD mice immunizedwith the p58, p61Y, p546, p550 or p883 peptide, by repeated stimulationin vitro with the decreasing concentrations (10 μM-1 μM) of the samepeptide.

The avidity of these lines for their inducer peptide was determined bymeasuring, as described above, their cytotoxicity with respect toRMAS-HHD target cells loaded with increasing concentrations (1 pM to 10μM) of the peptide concerned.

The results are given in FIG. 3.

These results show that the mCTL58, mCTL61Y, mCTL546, mCTL550 andmCTL883 lines have a relatively high avidity. 50% of maximum lysis isobtained for peptide concentrations which range from 3 nM in the case ofmCTL546 to 40 nM in the case of mCTL61Y.

EXAMPLE 3 Recognition of the Naturally Processed Epitopes of the EphA2Antigen by CTLs Induced by the p58 or p550 Peptides

To test whether the p58 and p550 peptides constitute naturally processedepitopes of the EphA2 antigen, the response of the cells of the mCTL58and mCTL550 lines to cells expressing this antigen was evaluated in twodifferent ways.

1) Stimulation with Transfected COS-7 Cells Expressing EphA2

The cells of the mCTL58 and mCTL550 lines are stimulated with monkeyCOS-7 cells cotransfected with the HHD construct (Pascolo et al.,mentioned above) and a plasmid containing the EphA2 cDNA. COS-7 cellstransfected either with the HHD construct alone or with the plasmidcontaining the EphA2 cDNA alone are used as negative controls.

The CTL stimulation is evaluated by measuring their TNF-α secretion. TheCOS-7 cells transfected with the HHD construct and loaded with the p58or p550 peptide are used as a positive control.

Four days after transfection, the COS-7 cells are brought into contactwith the mCTL58 and mCTL550 cells in a proportion of 5×10⁴ CTLs per3×10⁴ COS-7 cells, in RPMI 1640 in the presence of 10% SVF.

After incubation for 6 hours, the supernatant is removed (50 μl) andbrought into contact with WEHI164 clone 13 mouse fibrosarcoma cells(3×10⁴ per well), which are characterized by a high sensitivity toTNF-α-induced apoptosis. In order to quantify the TNF content in theculture supernatant, a standard range of TNF-α (concentrations of 0 to10⁴ pg/ml) is used in parallel. After incubation at 37° C. for 16 hours,the viability of the WEHI-164 clone 13 cells is determined by means ofan MTT colorimetric assay (SIGMA) (Espevik and Nissen Meyer, J. Immunol.Methods., 95, 99, 1986).

The results are given in FIG. 4.

-   -: untransfected COS-7 cells;-   EphA2: COS-7 cells transfected with the EphA2 cDNA alone;-   HHD: COS-7 cells transfected with the HHD construct alone;-   HHD+peptide: COS-7 cells transfected with the HHD construct and    loaded with the p58 or p550 peptide;-   HHD+EphA2: COS-7 cells transfected with the HHD construct and the    EphA2 cDNA.

These results show that the mCTL58 and mCTL550 lines respond tostimulation with the COS cells coexpressing HHD and EphA2.

On the other hand, no response is observed to the COS cells transfectedseparately with the HHD construct or with the EphA2 cDNA.

2) Stimulation with HLA-A*0201 Human Tumor Cells Expressing EphA2

The following HLA-A*0201 tumor lines were used: SAOS (sarcoma), 1355(lung cancer), Caco-2 (colon cancer), HIEG (renal carcinoma), LNCaP(prostate cancer). The DU145 line (prostate cancer), which does notexpress HLA-A*0201, was also used as a negative control.

Among these lines, DU145 and Caco-2 are known to express EphA2, andLNCaP is known not to express EphA2.

The expression of EphA2 in the other tumor lines was evaluated byWestern blotting. The level of EphA2 expression in all the lines used issummarized in Table II below. TABLE II HLA-A*0201 Cell line expressionEphA2 expression SAOS + + 1355 + + Caco-2 + + HIEG + + LNCaP + − DU145 −++: strong expression−: no expression.

The mCTL58 and mCTL550 lines were stimulated with the SAOS, 1355,Caco-2, HIEG, LNCaP and DU145 tumor lines mentioned above. The mCTL61Y,mCTL546 and mCTL 883 lines were stimulated with the LNCaP, DU145 andCaco-2 tumor lines mentioned above. The stimulation is evaluated by thedetection of TNF-α secretion as described above.

The results are given in FIGS. 5A, 5B and 5C.

FIG. 5A shows that the mCTL58 and mCTL550 cells respond to thestimulation with the Caco-2 cells, which express HLA-A*0201 and EphA2,but respond neither to the DU145 cells, which do not express HLA-A*0201,nor to the LNCaP cells, which do not express EphA2.

FIG. 5B shows that the mCTL58 and mCTL550 cells respond to thestimulation with the HIEG, Caco-2, 1355 and SAOS cells, which expresslarge amounts of EphA2, but do not respond to the LNCaP cells, which donot express EphA2.

FIG. 5C shows that the mCTL61Y, mCTL546 and mCTL883 cells respond to thestimulation of the Caco-2 cells, which express large amounts of EphA2,but do not respond to the LNCaP or DU145 cells, which do not expressEphA2 and HLA-A*0201, respectively.

The results of the above experiments show that the CTLs induced by p58,p61Y, p546, p550 or p883 recognize naturally processed epitopes of theEphA2 antigen.

EXAMPLE 4 Induction of Human CTLs Specific for the p58 or p550 Peptides

The ability of p58 and p550 to induce CTLs in vitro from peripheralblood mononuclear cells (PMBCs) from normal donors was tested asfollows.

The PBMCs are obtained, from blood samples taken by leukocytapheresis onnormal donors, after centrifugation at 2000 rpm for 20 min on aFicoll/Hypaque (Amersham) gradient. After 3 washes in 0.9% NaCl, 10⁷PBMCs are resuspended in each of the wells of a 6-well culture plate, in3 ml of complete medium (RPMI 1640 supplemented with 10% ofheat-inactivated human AB serum), and incubated at 37° C. for 2 hours.After incubation, the cells which have not adhered are removed and thecells which have adhered are differentiated into dendritic cells byadding to each of the wells 3 ml of complete medium containing 50 ng/mlof GM-CSF (R & D Systems), and 1000 IU/ml of IL-4 (R & D Systems). After7 days of culture, the dendritic cells are collected and loaded with thep58 or p550 peptide by incubation for 4 hours at 20° C. with 40 μg/ml ofpeptide in the presence of 3 μg/ml of β2-microglobulin, and are thenirradiated at 4200 rads; they are subsequently washed in order to removethe free peptide. CD8+ cells are isolated from the non-adherent cells bymeans of microbeads coupled to an anti-CD8 antibody (Miltenyi Biotec).

0.5×10⁶ CD8+ cells are stimulated by coculture in a 48-well plate with2.5×10⁴ dendritic cells loaded with the p58 or p550 peptide, in completemedium supplemented with 10 ng/ml of IL-7, in a final volume of 500μl/well. On the day after the placing of the cells in culture, 10 ng/mlof human IL-10 (R & D Systems) are added to each of the wells; on thesecond day, 30 IU/ml of human IL-2 are added to each of the wells. Ohthe seventh and the fourteenth day after the first stimulation, the CD8+cells are restimulated with the adherent cells loaded with 10 μg/ml ofpeptide in the presence of 3 μg/ml of β2-microglobulin, and irradiated.IL-10 (10 ng/ml) and IL-2 (30 IU/ml) are added 24 hours and 48 hours,respectively, after restimulation. Seven days after the secondrestimulation, the response of these cells to T2 cells loaded with p58or P550 or with an irrelevant peptide, or to Caco-2 HLA-A*0201 tumorcells (expressing EphA2 and HLA-A*0201), LNCaP HLA-A*0201 tumor cells(expressing HLA-A*0201 but not expressing EphA2) and DU145 tumor cells(expressing EphA2 but not expressing HLA-A*0201), is evaluated byassaying the intracellular IFNγ production.

The hCTL58 or hCTL550 cells are incubated with the loaded T2 cells, orwith the cells of the tumor line tested, in the presence of 20 μg/ml ofBrefeldin-A (Sigma). After 6 hours, they are washed, labeled with ananti-CD8 antibody conjugated to r-phycoerythrin (Caltag Laboratories) inPBS for 25 min at 4° C., washed and fixed with 4% paraformaldehyde. Theyare then permeabilized with saponin (Sigma) at 0.2% in PBS, and labeledwith an anti-IFNγ monoclonal antibody conjugated to allophycocyanin(Pharmingen).

The cells are then analyzed by flow cytometry (FACSCalibur™ (BectonDickinson) and the CellQuest™ program).

The results (expressed as number of CD8+ cells producing IFNγ per 10⁵CD8+ cells, are given in FIGS. 6A and 6B.

FIG. 6A shows that the human CTLs obtained from CD8+ cells stimulated,respectively, with the p58 peptide (hCTL58) or the p550 peptide(hCTL550) are activated by the T2 cells loaded with the correspondingpeptide, and that no activation by the T2 cells loaded with theirrelevant peptide is observed.

FIG. 6B shows a response of the hCTL58 and hCTL550 CTLs with respect tothe Caco-2 tumor line (EphA2⁺, HLA-A*0201⁺), but not with respect to theLNCaP (EphA2⁻, HLA-A*0201⁺) and DU145 (EphA2⁺, HLA-A*0201⁻) lines.

These results demonstrate that the p58 or p550 peptides induce humanCTLs capable of recognizing HLA-A*0201+tumor cells expressing EphA2.

1. An immunogenic peptide constituting a T epitope presented by MHC I,characterized in that it consists of a fragment of 8 to 11 consecutiveamino acids of the EphA2 antigen.
 2. The immunogenic peptide as claimedin claim 1, characterized in that it is chosen from: the peptide ofsequence IMNDMPIYM (SEQ ID NO: 4); the peptide of sequence VLLLVLAGV(SEQ ID NO: 6); the peptide of sequence VLAGVGFFI (SEQ ID NO: 7); thepeptide of sequence TLADFDPRV (SEQ ID NO: 8).
 3. An immunogenic peptideconstituting a T epitope presented by MHC I, characterized in that it isderived from a peptide consisting of a fragment of 8 to 11 consecutiveamino acids of the EphA2 antigen, by substitution of at least one aminoacid of said peptide with an amino acid which increases the affinity ofsaid peptide for an MHC I allele.
 4. The peptide as claimed in claim 3,characterized in that it is derived from a peptide consisting of afragment of 8 to 11 consecutive amino acids of the EphA2 antigen, bysubstitution of the N-terminal amino acid of said peptide with atyrosine residue.
 5. The peptide as claimed in claim 4, characterized inthat it is defined by the sequence YMPIYMYSV (SEQ ID NO: 9).
 6. Apolynucleotide encoding a peptide as claimed in any one of claims 1 to5.
 7. A composition comprising at least one peptide as claimed in anyone of claims 1 to 6 or a polynucleotide as claimed in claim
 5. 8. Thecomposition as claimed in claim 7, characterized in that it is amultiepitope composition also comprising one or more other immunogenicpeptide(s) or one or more polynucleotide(s) encoding said peptide(s). 9.The composition as claimed in claim 7, characterized in that it is achimeric polypeptide comprising at least one copy of a peptide asclaimed in any one of claims 1 to 5 and/or at least one copy of anotherimmunogenic peptide, or a polynucleotide encoding said chimericpolypeptide.
 10. The use of a peptide as claimed in any one of claims 1to 5, of a polynucleotide as claimed in claim 6 or of a composition asclaimed in any one of claims 7 to 9, for obtaining a medicinal product.11. The use as claimed in claim 10, characterized in that said medicinalproduct is intended for antitumor immunotherapy.
 12. The use as claimedin claim 11, characterized in that said medicinal product is intendedfor the immunotherapy of tumors expressing the EphA2 antigen.
 13. Theuse as claimed in any one of claims 10 to 12, characterized in that saidmedicinal product is intended for the treatment of HLA-A*0201 patients.